Pesticide resistance in disease-carrying arthropods, important to agriculture, veterinary science, and public health, poses a serious threat to global vector control programs. Previous studies have shown that blood-sucking arthropod vectors suffer high mortality when ingesting blood containing inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD, the second enzyme in the tyrosine metabolic pathway). This study examined the efficacy of HPPD inhibitors in β-triketone herbicides against three major mosquito vector species, including those transmitting traditional diseases such as malaria, emerging infectious diseases such as dengue fever and Zika virus, and emerging viral threats such as oropuche virus and ursutu virus. These species included both pyrethroid-susceptible and pyrethroid-resistant mosquitoes.
Only nitisidone (not mesotrione, sulfadiazine, or thiamethoxam) exhibited significant mosquito control activity when blood-sucking mosquitoes came into contact with treated surfaces. No significant difference in susceptibility to nitisidone was found between insecticide-sensitive Anopheles gambiae mosquitoes and mosquito strains with multiple resistance mechanisms. The compound demonstrated consistent efficacy against all three mosquito species tested, indicating broad-spectrum activity against major disease vectors.
This study demonstrates that nitisidone has a novel mechanism of action, distinct from existing Insecticide Resistance Action Committee (IRAC) classifications, targeting the blood digestion process. Nitisidone’s efficacy against resistant strains and its potential for integration with existing vector control measures, such as treated mosquito nets and indoor insecticide spraying, make it an ideal candidate for expanding prevention and control strategies for malaria, dengue fever, Zika virus disease, and other emerging viral diseases.
Interestingly, standard World Health Organization bioassays use only sugar-fed mosquitoes to test discriminant concentrations of insecticides that may be non-lethal to blood-sucking mosquitoes.[38] This highlights the importance of considering potential differences in effective doses between blood-sucking and non-blood-sucking mosquitoes, which may influence residual efficacy and resistance development. Although discriminant doses (DDs) are typically determined based on LD99 values for blood-sucking mosquitoes, differences in insect physiology can influence their susceptibility, and therefore testing only blood-sucking mosquitoes may not fully reflect the range of resistance levels.
This study focused on the efficacy of three mosquito species—Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus—in a bloodsucking test, which simulates mosquito landing on a wall and serves as a target for indoor treatment with long-lasting insecticides (IRS). All female mosquitoes were killed upon contact with nitisidone-coated surfaces, but not with other HPPD β-triketone inhibitors. Leveraging the uptake of HPPD inhibitors by mosquito legs represents a promising strategy for overcoming insecticide resistance and improving vector control. This study supports the need for further research and development of nitisidone for indoor treatment with long-lasting insecticides as an alternative to existing insecticidal sprays.
Three methods for assessing the efficacy of nitisidone as an external insecticide were compared. Differences were analyzed between tests using topical application, insect leg application, and bottle application, as well as the application method, insecticide delivery method, and exposure time.
However, despite the difference in mortality rates between New Orleans and Mukhza at the highest dose, all other concentrations were more effective in New Orleans (susceptible) than in Mukhza (resistant) after 24 hours.
To explore innovative vector control strategies, a promising approach to discovering new insecticidal compounds is to expand research beyond traditional targets of the nervous system and detoxification genes to include insect bloodsucking mechanisms. Previous studies have shown that nitisidone is toxic following ingestion by bloodsucking insects or after epidermal absorption following topical application (using a solvent) .
Integrating data from multiple detection methods can improve the reliability of insecticide efficacy assessments. However, it should be noted that of the three methods considered, the topical application method is the least representative of real field conditions. Direct application of insecticides to the thorax of mosquitoes using an aqueous solution does not mimic typical exposure to Anopheles gambiae sl. [47], although it may provide an approximate indication of Anopheles susceptibility to a particular compound. Although both the glass plate and bottle methods measure bioactivity through leg contact, their results are not directly comparable. Differences in exposure time and surface coverage can significantly influence the mortality observed with each detection method; therefore, choosing an appropriate detection method is critical for accurately assessing insecticide efficacy.
Residual-effect insecticide (RIA) spraying exploits the post-feeding resting behavior of mosquitoes, causing them to ingest insecticides upon contact with treated surfaces. Insecticide degradation, insufficient spray coverage, and handling of treated surfaces (e.g., washing walls after treatment) can significantly reduce the effectiveness of RIA. These issues lead to two difficulties: (1) mosquitoes can survive exposure to non-lethal doses; and (2) although resistance is primarily driven by lethal selection, repeated exposure to sublethal doses can promote the evolution of resistance by allowing some resistant individuals to survive and maintaining alleles associated with reduced susceptibility [54]. Because we used blood-feeding mosquitoes instead of industry-standard sugar-feeding mosquitoes, direct comparison with previously published data was not possible. However, a comparison of the discriminant dose (DD) and the dose-response curve shape of nitisidone with data for other compounds [47] is encouraging. The discriminant dose combines a fixed exposure time and the amount of insecticide applied to the vial, with the amount of adsorbed compound depending on the actual contact time on the paw. Based on these results, nitisidone is more potent than thiamethoxam, spinosad, mefenoxam, and dinotefuran [47], making it an ideal candidate for new indoor insecticide formulations that require further optimization. Considering the slope of the dose-response curve (which was approximated by calculating the LC95 and LC50 slopes in Figure 3), nitisidone had the steepest curve, indicating its high efficacy. This is consistent with previous studies of nitisidone in blood-feeding and topical tests on another dipteran vector, the tsetse fly (Glossina morsitans morsitans) [26]. We briefly tested the efficacy of nitisidone (using a glass plate test) by exposing Kissou mosquitoes (Figure S1A) or New Orleans mosquitoes (Figure S1B) to nitisidone before feeding. Nitisidone remained effective on the legs, simulating the scenario of mosquitoes landing on a wall treated with nitisidone before feeding, which requires further investigation. The efficacy of nitisidone (and other HPPD inhibitors) on the legs may be enhanced by combination with adjuvants such as rapeseed methyl ester (RME), as described for other insecticides [44, 55]. By testing the effects of RME on *Gnaphalium affine* before feeding (Figure S2), we found that at a concentration of 5 mg/m², the combination with adjuvants such as RME significantly increased mosquito mortality.
The kinetics of mosquito killing by unformulated nitisidone in various resistant strains is of interest. The slower mortality of the VK7 2014 strain may be due to thickened epidermis, reduced blood consumption, or accelerated blood digestion—factors we did not investigate. Nitisidone showed low toxicity to the resistant Culex muheza mosquito strain, suggesting the need for further studies at higher concentrations (25 to 125 mg/m²). Furthermore, similar to Culex, Aedes mosquitoes are less sensitive to nitisidone than Anopheles, which may indicate physiological differences between the two species in terms of blood consumption and digestion rate [27]. These differences highlight the importance of understanding species-specific characteristics when evaluating blood-activated insecticides. Despite its blood-dependent and delayed action, nitisidone may have practical value because it can act before mosquitoes lay eggs or reduce their overall fecundity. Due to its unique mechanism of action, targeting the tyrosine degradation pathway by inhibiting 4-hydroxyphenylpyruvate dioxygenase (HPPD), nitisidone holds promise as part of a comprehensive vector control strategy. However, the possibility of developing drug resistance due to mutations in the target site or metabolic adaptations must be considered, and further research is currently underway to explore these mechanisms.
Our results demonstrate that nitisidone kills blood-sucking mosquitoes via leg contact, a mechanism not observed with mesotrione, sulfadiazine, and thiamethoxam. This killing effect does not discriminate between mosquito strains sensitive or highly resistant to other classes of insecticides, including pyrethroids, organochlorides, and potential carbamates. Furthermore, the epidermal absorption efficiency of nitisidone is not limited to Anopheles species; this is confirmed by its efficacy against Culex pipiens pallens and Aedes aegypti. Our data support the need for further research to optimize nitisidone absorption, for example, by chemically enhancing epidermal absorption or using adjuvants. Through its unique mechanism of action, nitisidone effectively exploits the blood-sucking behavior of female mosquitoes. This makes it an ideal candidate for innovative indoor insecticidal sprays and mosquito nets with long-lasting insecticidal action, especially in areas where traditional mosquito control methods are weakened by the rapid spread of pyrethroid resistance.
Post time: Dec-23-2025